The invention relates to nucleic acid sequences which regulate expression of a nucleotide sequence of interest. In particular, the invention relates to nucleic acid sequences which regulate expression of a nucleotide sequence of interest in an age-related manner and/or in a liver-specific manner. The invention further relates to methods of using the regulatory nucleic acid sequences provided herein for age-related and/or liver-specific expression of nucleotides sequences of interest. The invention also relates to host cells and to transgenic non-human animals which harbor the regulatory nucleic acid sequences of the invention. The compositions and methods of the invention are useful in regulating expression of a nucleotide sequence of interest in an age-related and/or liver-specific manner.
A multitude of human diseases (e.g., thrombosis, cardiovascular diseases, diabetes, Alzheimer""s disease, cancer, osteoporosis, osteoarthritis, Parkinson""s disease, dementia) are associated with increasing age and result in serious effects on the quality of life and on the life expectancy of individuals suffering from such diseases. Other diseases (e.g., cirrhosis, primary and metastatic neoplasia, Wilson disease, hepachromatosis, infectious hepatitis, hepatic necrosis, Gilbert disease, Criggler-Najar disease) which afflict the liver also have serious clinical manifestations and are responsible for high morbidity and mortality.
The treatment of age-related diseases (i.e., diseases whose prevalence and/or severity of clinical manifestations increases with the age of the patient) and diseases afflicting the liver focuses on the alleviation of the general symptoms of the disease using one or a combination of two modalities, i.e., non-pharmacological treatment and pharmacological treatment. Non-pharmacological treatment include, for example, periods of bed rest and dietary changes. Non-pharmacological treatment is often used as an adjunct to pharmacological treatment which involves the use of drugs. Unfortunately, many of the commonly used pharmacological agents have numerous side effects and their use is further exacerbated by the non-responsiveness by many patients with severe disease, who, paradoxically, are in most need of treatment. Both non-pharmacological and pharmacological treatments provide unsatisfactory approaches to treating age-related and liver-associated diseases because these approaches are often ineffective, their effects are inconsistent, and are directed to alleviating the general symptoms of disease, rather than to specifically addressing the source of morbidity and mortality. Moreover, no suitable animal models are currently available to rationally design drugs which target specific biochemical and physiological pathways which are associated with age-related and with liver-associated diseases.
What is needed are methods for age-related and liver-specific gene expression and models for age-related and liver-specific diseases.
The invention provides nucleic acid sequences which regulate expression of a nucleotide sequence of interest in an age-related manner, as well as nucleic acid sequences which direct liver-specific expression of a gene of interest. Further provided by the invention are transgenic animals which may be used as models for age-related and/or liver specific diseases.
In one embodiment, the invention provides a recombinant expression vector comprising in operable combination i) a nucleic acid sequence of interest, ii) a promoter sequence, and iii) one or more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3. Without intending to limit the invention to any particular type or source of nucleic acids sequence of interest, in a preferred embodiment, the nucleic acid sequence of interest encodes a protein selected from factor VIII, factor VII, factor IX, factor X, prothrombin, protein C, antithrombin III, tissue factor pathway inhibitor, LDL-receptor, human xcex11-antitrypsin, antithrombin III, PEA-3 protein, xcex2-galactosidase, and luciferase. While it is not intended that the invention be restricted to any particular type or source of promoter sequence, in an alternative preferred embodiment, the promoter sequence is selected from human factor IX promoter, cytomegalovirus promoter, tRNA promoter, 5S rRNA promoters, histone gene promoters, RSV promoter, retrovirus LTR promoter, SV40 promoter, PEPCK promoter, MT promoter, SRxcex1 promoter, P450 family promoters, GAL7 promoter, T7 promoter, T3 promoter, SP6 promoter, K11 promoter, and HIV promoter. It is not contemplated that the invention be limited to any particular age regulatory sequence which is a portion of SEQ ID NO:1. However, in another preferred embodiment, the age regulatory sequence which is a portion of SEQ ID NO:1 is selected from SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. Without intending to limit the invention to any particular rage regulatory sequence which s a portion of SEQ ID NO:3, in yet another preferred embodiment, the age regulatory sequence which is a portion of SEQ ID NO:3 is selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.
Also provided by the invention is a host cell containing a recombinant expression vector comprising in operable combination i) a nucleic acid sequence of interest, ii) a promoter sequence, and iii) one or more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3. Without intending to limit the invention to the environment in which the host cell is contained, in one preferred embodiment, the host cell is comprised in a tissue or organ in a living animal. In another preferred embodiment, the host cell is a gamete. In yet another preferred embodiment, the host cell is selected from bacterial cell, yeast cell, plant cell, insect cell, and mammalian cell.
The invention also provides a recombinant expression vector comprising in operable combination i) a nucleic acid sequence of interest, ii) a promoter sequence, and iii) a functional homolog of one or more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3. Without limiting the invention to the type or source of the nucleic acid sequence of interest, in one preferred embodiment, the nucleic acid sequence of interest encodes a protein selected from factor VIII, factor VII, factor IX, factor X, prothrombin, protein C, antithrombin III, tissue factor pathway inhibitor LDL-receptor, human xcex11-antitrypsin, antithrombin III, PEA-3 protein, xcex2-galactosidase, and luciferase. While it is not intended that the invention be limited to the type or source of the promoter sequence, in an alternative preferred embodiment, the promoter sequence is selected from human factor IX promoter, cytomegalovirus promoter, tRNA promoter, 5S rRNA promoters, histone gene promoters, RSV promoter, retrovirus LTR promoter, SV40 promoter, PEPCK promoter, MT promoter, SRxcex1 promoter, P450 family promoters, GAL7 promoter, T7 promoter, T3 promoter, SP6 promoter, K11 promoter, and HIV promoter. Though it is not contemplated that the invention be limited to the portion of SEQ ID NO:1 which has age-related regulatory activity, in another preferred embodiment, the age regulatory sequence which is a portion of SEQ ID NO:1 is selected from SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. Without intending to limit the invention the portion of SEQ ID NO:3 which has age-related regulatory activity, in yet another preferred embodiment, the age regulatory sequence which is a portion of SEQ ID NO:3 is selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.
Also provided herein is a host cell containing recombinant expression vector comprising in operable combination i) a nucleic acid sequence of interest, ii) a promoter sequence, and iii) a functional homolog of one or more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3. Without intending to limit the invention to the environment in which the host cell is contained, in one preferred embodiment, the host cell is comprised in a tissue or organ in a living animal. In an alternative preferred embodiment, the host cell is a gamete. In another preferred embodiment, the host cell is selected from bacterial cell, yeast cell, plant cell, insect cell, and mammalian cell.
The invention also provides a method, comprising: a) providing: i) a cell, ii) a nucleic acid sequence of interest, iii) a promoter sequence, and iv) one or more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest, the promoter sequence, and the one or more age regulatory sequences to produce a transgene; and c) introducing the transgene into the cell to create a treated cell under conditions such that the nucleic acid sequence of interest is expressed in the treated cell. Without intending to limit the treated cell to any particular environment, in one preferred embodiment, the treated cell is comprised in a tissue or organ in a living animal.
The invention further provides a substantially purified nucleic acid sequence comprising a nucleotide sequence selected from a functional homolog of SEQ ID NO:1 and of the complement thereof.
Also provided herein is a substantially purified nucleic acid sequence comprising a nucleotide sequence selected from a functional homolog of SEQ ID NO:3 and of the complement thereof.
Also provided by the present invention is a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:1 and of the complement thereof. In one embodiment, the portion is SEQ ID NO:2. In an alternative embodiment, the portion is selected from SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
The invention also provides a substantially purified nucleic acid sequence comprising a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:3 and of the complement thereof. In one embodiment, the portion is selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.
Also provided herein is a substantially purified nucleic acid sequence which hybridizes under stringent hybridization conditions with SEQ ID NO:1 or with the complement thereof, wherein the nucleic acid sequence is characterized by having age-related regulatory activity, and by having greater than 63% and less than 100% homology to the SEQ ID NO:1.
The invention also provides a substantially purified nucleic acid sequence which hybridizes under stringent hybridization conditions with SEQ ID NO:3 or with the complement thereof, wherein the nucleic acid sequence is characterized by having age-related regulatory activity, and by having greater than 60% and less than 100% homology to the SEQ ID NO:3.
The invention additionally provides a recombinant expression vector comprising at least a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:1 and of the complement thereof.
Also provided herein is a recombinant expression vector comprising at least a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:3 and of the complement thereof.
The invention also provides a transgenic cell comprising at least a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:1 and of the complement thereof. In one embodiment, the nucleotide sequence is operably linked to a promoter and to a nucleic acid sequence of interest. In a preferred embodiment, the transgenic cell is comprised in an animal. In a more preferred embodiment, the nucleic acid sequence of interest is expressed in an age-related manner in the transgenic cell.
The invention additionally provides a transgenic cell comprising at least a portion of a nucleotide sequence selected from a functional homolog of SEQ ID NO:3 and of the complement thereof. In one embodiment, the nucleotide sequence is operably linked to a promoter and to a nucleic acid sequence of interest. In a preferred embodiment, the transgenic cell is comprised in an animal. In a more preferred embodiment, the nucleic acid sequence of interest is expressed in an age-related manner in the transgenic cell.
The invention also provides a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; iv) SEQ ID NO:1; and v) SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest, the promoter sequence, the SEQ ID NO:1 and the SEQ ID NO:3 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell. In one embodiment, the cell expresses a recombinant protein identified as SEQ ID NO:47. In an alternative embodiment, the cell is selected from HepG2 cell, fibroblast cell, myoblast cell, and endothelial cell. In another embodiment, the cell is a fertilized egg cell, and the transgenic cell is a transgenic fertilized egg cell. In a preferred embodiment, the method further comprises d) introducing the transgenic fertilized egg cell into a non-human animal and permitting the animal to deliver progeny containing the transgene. In a more preferred embodiment, the progeny is characterized by age-related expression of the nucleic acid sequence of interest. In an alternative more preferred embodiment, the progeny is characterized by liver-specific expression of the nucleic acid sequence of interest. In another preferred embodiment, the fertilized egg cell is derived from a mammal of the order Rodentia. In a more preferred embodiment, the fertilized egg cell is a mouse fertilized egg cell. In yet another embodiment, the promoter is selected from human factor IX promoter, cytomegalovirus promoter, tRNA promoter, 5S rRNA promoters, histone gene promoters, RSV promoter, retrovirus LTR promoter, SV40 promoter, PEPCK promoter, MT promoter, SRxcex1 promoter, P450 family promoters, GAL7 promoter, T7 promoter, T3 promoter, SP6 promoter, K11 promoter, and HIV promoter. In a further embodiment, the nucleic acid sequence of interest encodes a protein selected from factor VIII, factor VII, factor IX, factor X, prothrombin, protein C, antithrombin III, tissue factor pathway inhibitor, LDL-receptor, human xcex11-antitrypsin, antithrombin III, PEA-3 protein, xcex2-galactosidase, and luciferase.
The invention also provides a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; iv) a portion of SEQ ID NO:1; and v) a portion of SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest, the promoter sequence, the portion of SEQ ID NO:1 and the portion of SEQ ID NO:3 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell.
Additionally provided by the invention is a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; and iv) SEQ ID NO:1; b) operably linking the nucleic acid sequence of interest, the promoter sequence, and the SEQ ID NO:1 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell.
Also provided herein is a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; and iv) a portion of SEQ ID NO:1; b) operably linking the nucleic acid sequence of interest, the promoter sequence, and the portion of SEQ ID NO:1 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell.
The invention further provides a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; and iv) SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest, the promoter sequence, and the SEQ ID NO:3 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell.
Further provided by the invention is a method for expressing a nucleic acid sequence of interest in a cell, comprising: a) providing: i) a cell; ii) a nucleic acid sequence of interest; iii) a promoter sequence; and iv) a portion of SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest, the promoter sequence, and the portion of SEQ ID NO:3 to produce a transgene; and c) introducing the transgene into the cell to produce a transgenic cell under conditions such that the nucleic acid sequence of interest is expressed in the transgenic cell.